PROJECT SUMMARY/ABSTRACT Craniofacial FD is one of the most common and debilitating skeletal dysplasias. The expansile, fibrous bony lesions that form in the facial bones and skull base cause significant disability, including dysmorphic facies, bone fragility, pain, and vision and hearing loss. There are no effective medical treatments for FD, making this an area of critical medical need. FD can develop in one bone (monoostotic) or multiple bones (polyostotic) and can occur in association with McCune-Albright Syndrome (MAS), which is a somatic mosaic genetic disease characterized by FD, precocious puberty, caf-au-lait skin lesions, various endocrinopathies, and solid organ malignancies. FD is arguably the most clinically significant feature of MAS. FD can form at any site within the skeleton, but the most commonly involved sites are the neural crest-derived craniofacial bones. Despite this, little is known about the cellular and molecular mechanisms that drive craniofacial FD, and why there is a predilection for FD formation in neural crest-derived bone. In addition to craniofacial FD, patients with MAS also commonly have mosaic involvement of other neural crest-derived tissues, including melanocytic skin lesions and pituitary and adrenal tumors. The goal of this proposal is to better understand the cellular and molecular mechanisms that drive craniofacial FD so that we can work towards identifying potential treatment targets. We will pursue this goal through the following 3 aims: Aim 1: Test if WNT inhibition reverses craniofacial FD in Col1(2.3)+/Rs1+ mice. We have previously modeled FD in mice by activating Gs-GPCR signaling in osteoblastic cells via the engineered GPCR ?Rs1.? Col1(2.3)+/Rs1+ mice develop a dramatic bone phenotype that resembles human FD. We have shown that stopping the abnormal Gs-GPCR signaling reverses the FD lesions, suggesting that FD can be reversed. We also found increased levels of WNT expression in bones harvested from these mice, suggesting that WNT signaling is important in FD. We will use this model to test if inhibition of WNT signaling can reverse craniofacial FD lesions. Aim 2: Determine the GNASR201H mutational burden in human craniofacial FD bone and identify the cell type(s) that harbor this mutation. Despite knowing the causative mutation in FD/MAS, we still do not know which cell type(s) carry the mutation in human FD bone and what level of mosaicism is needed in order to cause disease. We will use an innovative approach to isolate and genotype single cells from human craniofacial FD lesions. This will allow us to understand the mutational burden of human FD lesions and identify the cell type(s) that carry the mutation. Aim 3: Elucidate how the mosaic GNASR201H mutation affects cell multipotency and differentiation capacity using a human iPSC model of FD/MAS. We will use our newly-developed human iPSC model of MAS that contains the causative GNAS mutation in its endogenous locus to explore the effects of GNAS on early cell fate, particularly the neural crest and osteoblast lineages. Together, these aims seek to address the critical knowledge gaps that exist in our understanding of the pathogenesis of craniofacial FD.